N terminus of CtIP is critical for homologous recombination-mediated double-strand break repair

Abstract

DNA double-strand breaks (DSBs) represent one of the most lethal types of DNA damage cells encounter. CtIP (also known as RBBP8) acts together with the MRN (MRE11-RAD50-NBS1) complex to promote DNA end resection and the generation of single-stranded DNA, which is critically important for homologous recombination repair. However, it is not yet clear exactly how CtIP participates in this process. Here, we demonstrate that besides the known conserved C terminus, the N terminus of CtIP protein is also required in DSB end resection and DNA damage-induced G(2)/M checkpoint control. We further show that both termini of CtIP can interact with the MRN complex and that the N terminus of CtIP, especially residues 22-45, binds to MRN and plays a critical role in targeting CtIP to sites of DNA breaks. Collectively, our results highlight the importance of the N terminus of CtIP in directing its localization and function in DSB repair.

FIGURE 1.

Both the N and C termini of CtIP are required for G₂/M checkpoint control and HR repair following DNA damage. A, diagram of internal deletion mutants of siRNA-resistant FLAG-tagged CtIP constructs used in this study. FL, full-length CtIP; CC, coiled-coil domain. B, deletion of the N or C terminus of CtIP disrupts its function in G₂/M checkpoint control. HeLa cells stably expressing siRNA-resistant wild-type CtIP or internal deletion mutants were generated. These cells were transfected twice with CtIP siRNA, and G₂/M checkpoint assays were performed as described under “Experimental Procedures.” The percentage of phosphohistone H3 (pH3)-positive cells was determined by fluorescence-activated cell sorter analysis. Means ± S.D. (error bars) were obtained from three independent experiments. C, N and C termini of CtIP are required for CHK1 activation. The stable cells shown in B were transfected with control (siCtrl) or CtIP (siCtIP) siRNA. Forty-eight hours later, cells were treated with IR radiation (10 Gy) and allowed to recover for 1 h. Cells were collected and lysed. Immunoblotting was performed using anti-phospho-CHK1 (Ser³¹⁷), anti-CHK1, or anti-FLAG antibody. WT, wild-type CtIP. D, N and C termini of CtIP are required for RPA focus formation. Cells were transfected with siRNAs as described for C, irradiated (10 Gy), and allowed to recover for 6 h before fixation. Immunostaining was performed using the indicated antibodies. Scale bar = 10 μm. DAPI, 4′,6-diamidino-2-phenylindole. E, deletion of the N or C terminus of CtIP impairs HR repair. Cells were electroporated with DR-GFP and pCBASce plasmids (see “Experimental Procedures”). The percentage of GFP-positive cells was determined by flow cytometry 48 h after electroporation. The data were normalized to those obtained from cells transfected with control siRNA (set as 1.0). Means ± S.D. (error bars) were obtained from three independent experiments.

FIGURE 2.

Both the N and C termini of CtIP can interact with the MRN complex. A, CtIP can bind to all three components in the MRN complex. IP, immunoprecipitation. B, schematic representation of the Myc-tagged CtIP mutants used in this study. FL, full-length CtIP; CC, coiled-coil domain. C–E, N and C termini of CtIP can interact with NBS1, MRE11, or RAD50. 293T cells were transfected with plasmids encoding Myc-tagged wild-type CtIP (WT) or the N160, C166, N160C166, or 161–731 mutant of CtIP, together with plasmids encoding SBP-FLAG-tagged NBS1 (C), MRE11 (D), or RAD50 (E). Cells were lysed 24 h after transfection. Immunoprecipitation was carried out using S protein beads, and immunoblotting was performed using the antibodies indicated. F, N and C termini of CtIP can bind directly to MRN components. The in vitro binding assays described for A and F were performed using the baculovirus expression system. Sf9 cells were co-infected with baculoviruses expressing the indicated constructs. Forty-eight hours later, cells were collected and lysed, and pulldown experiments were performed using S protein beads or glutathione-agarose beads. Protein bound to the beads was eluted and resolved by SDS-PAGE and immunoblotting using the indicated antibodies.

FIGURE 3.

N terminus of CtIP is essential for its focus formation following DNA damage. A, the D1 mutant of CtIP (with residues 18–159 deleted) fails to form foci upon DNA damage. HeLa cells stably expressing FLAG-tagged wild-type (WT) or deletion mutant CtIP were seeded on coverslips overnight. Cells were irradiated with 10 Gy and allowed to recover for 4 h before fixation and immunostaining. DAPI, 4′,6-diamidino-2-phenylindole. B, schematic representation of FLAG-tagged CtIP mutants with smaller internal deletions at their N termini. CC, coiled-coil domain. C, residues 22–45 at the N terminus of CtIP are required for CtIP focus formation. Cell treatment and immunostaining were performed as described for B. Scale bars = 10 μm.

FIGURE 4.

Residues 22–45 of CtIP are required for NBS1 binding and damage-induced G₂/M checkpoint control. A, residues 22–45 of CtIP are required for binding between the N terminus of CtIP and NBS1. 293T cells were transfected with plasmids encoding Myc-tagged wild-type CtIP (WT) or N160 and N160 (d22–45) CtIP together with plasmids encoding SBP-FLAG-tagged NBS1. Cells were lysed 24 h after transfection. Immunoprecipitation (IP) was carried out using S protein beads, and immunoblotting was performed using the antibodies indicated. B, residues 22–45 of CtIP are required for CtIP function in G₂/M checkpoint control. HeLa cells stably expressing siRNA-resistant wild-type CtIP and deletion mutants were generated. These cells were transfected twice with CtIP siRNA, and G₂/M checkpoint assays were performed as described under “Experimental Procedures.” The percentage of phosphohistone H3 (pH3)-positive cells was determined by fluorescence-activated cell sorter analysis. Means ± S.D. (error bars) were obtained from three independent experiments. C, the stable cells shown in B were transfected with control (siCtrl) or CtIP (siCtIP) siRNA. Forty-eight hours later, cells were treated with IR radiation (10 Gy) and allowed to recover for 1 h. Cells were collected and lysed. Immunoblotting was performed using anti-phospho-CHK1 (Ser³¹⁷), anti-CHK1, or anti-FLAG antibody. D, proposed model of CtIP function in DNA damage repair.